1,2,3-triazole and chiral Schiff base hybrids as potential anticancer agents: DFT, molecular docking and ADME studies

A series of novel 1,2,3-triazole and chiral Schiff base hybrids 2–6 were synthesized by Schiff base condensation reaction from pre-prepared parent component of the hybrids (1,2,3-triazole 1) and series of primary chiral amines and their chemical structure were confirmed using NMR and FTIR spectroscopies, and CHN elemental analysis. Compounds 1–6 were evaluated for their anticancer activity against two cancer PC3 (prostate) and A375 (skin) and MRC-5 (healthy) cell lines by Almar Blue assay method. The compounds exhibited significant cytotoxicity against the tested cancer cell lines. Among the tested compounds 3 and 6 showed very good activity for the inhibition of the cancer cell lines and low toxicity for the healthy cell lines. All the compounds exhibited high binding affinity for Androgen receptor modulators (PDB ID: 5t8e) and Human MIA (PDB ID: 1i1j) inhibitors compared to the reference anticancer drug (cisplatin). Structure activity relationships (SARs) of the tested compounds is in good agreement with DFT and molecular docking studies. The compounds exhibited desirable physicochemical properties for drug likeness.


Results and discussions
The one component of the hybrids, 1,2,3-triazole 1 was Previously synthesized through multistep synthesis by copper catalyzed click chemistry reaction of preprepared 2-(prop-2-yn-1-yloxy) benzaldehyde with azidobenzene.The structure of the compound was confirmed by 1 H NMR spectroscopy.The disappearance of the signal for the terminal alkyne proton from 2-(prop-2-yn-1-yloxy) benzaldehyde at 2.55 ppm and appearance of the diagnostic low field singlet signal for the triazole ring proton at 8.08 ppm established the structure of 2-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (1) (Fig. S1) 44 .Other characterization techniques such as FTIR, mass spectrometry and CHN elemental analysis were performed which established the formation of 1 (see experimental procedure).Compound 1 was exposed to different solvents for obtaining single crystals suitable for single crystal X-ray diffraction.Single crystals of 1 was obtained after one week from ethanol by slow evaporation.Single crystal analysis (Fig. 2) revealed that 1 crystallized in triclinic space group Pī.Details of the X-ray crystallographic data for the compound are given in Table 1 and rest of the structural parameters (bond lengths/angles and hydrogen parameters) are provided in the supplementary documents (Table S1).The reaction for the synthesis of the hybrid compounds is shown in Fig. 3.The reaction was performed by Schiff base condensation of the preprepared triazole 1 with primary chiral amines (D-glutamic acid, L-tryptophan, L-tyrosine and L-histidine) and an enantiomer Phenylalanine ethyl ester hydrochloride in the presence of sodium hydroxide as a base and provided the proposed hybrids of 1,2,3-triazole and chiral Schiff bases (2-6).The structures of the compounds were characterized using NMR and FTIR spectroscopy (see supplementary material Figs.S1-S14) as well as CHN elemental analysis.
The 1 H NMR spectra of compounds (2-6) showed characteristic signals of the protons corresponding to the imine moiety between 9.02 and 8.06 ppm and the 1,2,3-triazole group between 8.92 and 7.92 ppm.The appearance of characteristic high field singlet signal between 5.55 and 2.68 ppm and between 5.26 and 2.63 ppm, respectively was attributed to the methine and methylene protons of the chiral amino acids.The appearance of characteristic high field singlet signal between 5.44 and 5.21 ppm was attributed to the methylene protons attached to the phenolic and 1,2,3-triazole groups.Moreover, the appearance of characteristic low field broad singlet signals in compounds 2 and 6 between 10.41 and 10.34 ppm and at 10.42 ppm, respectively was assigned to the hydroxyl protons of the chiral amino acids (D-glutamic acid and phenyl alanine).The broad singlet signal at 10.68 ppm in compound 3 is evident for the NH proton of the chiral amino acid (L-tryptophan).The absence of characteristic low field broad singlet signal in compounds 3, 4, and 5 is due to the deprotonation of the hydroxyl protons of the chiral amino acids by sodium hydroxide.In 13 C NMR spectra, the presence of characteristic signals between 160.4 and 157.0 ppm correspond to the imine carbon (C=N).The presence of characteristic signals between 144.3 and 143.3 ppm for N-C=CH and between 120.3 and 120.1 ppm for N-C=CH, correspond to the 1,2,3-triazole carbons.The carbonyl carbon is evident between 192.1 and 174.8 ppm.The characteristic signals between 98.6 and 59.7 ppm for N-CH-CH 2 and between 55.5 and 30.1 ppm for N-CH-CH 2, respectively correspond to the methine and methylene carbons of the chiral amino acids.The characteristic signal for the methylene carbon (CH 2 OAr) is attributed between 61.9 and 56.6 ppm.The molecular structures of compounds 2-6 were also characterized by FTIR spectroscopy (Fig. S14).The FTIR absorption spectra of compounds showed characteristic bands of the imine moiety between 1595 and 1598 cm −1 corresponding to the stretching of C=N bond.In compound 2, the broad absorption band at ca. 3479 cm −1 and the strong absorption peak at 1667 cm −1 were attributed to the υ (OH stretch) and υ (C=O stretch) of the glutamic acid, respectively.In compound 6, the broad absorption band at ca. 3353 cm −1 was attributed to the υ (OH stretch) of the phenyl alanine.The absence of characteristic broad absorption band for υ (OH stretch) in the FTIR spectra of compounds 3, 4, and 5 is evident for the deprotonation of the hydroxyl protons of the chiral amino acids by sodium hydroxide.Summary of 1 H and 13 C chemical shift of azomethine moiety, IR absorption of C=N and CHN elemental analysis results for compounds 2-6 is shown in Table 2.

Anticancer study
The previously synthesized parent component of the hybrids (1,2,3-triazole)  IC 50 values in the range of 76.90-93.07μg/mL better than the standard cisplatin which showed high toxicity at a concentration of 60.34 μg/mL.The test results for cytotoxicity and IC 50 values of compounds 1-6 treated against PC3 cancer cell lines at different concentrations 5, 15, 25, 50, 75 and 100 μg/mL is presented in Table S2.The cytotoxicity is concentration dependent and all the compounds except for 3 and 6 did not show any significant potency for the inhibition of the cancer cells in the concentration range of 5-50 μg/mL.All the compounds except for 3 did exhibit any significant activity in the concentration range of 5-25 μg/mL for the inhibition of A375 cancer cell lines (Table S3).Based on IC 50 values of tested compounds, the order of cytotoxicity is 3 > 6 > 4 > 5 > 2 > 1 and this revealed that hybridisation of 1 with the series of chiral amines played a vital role for the activity.Among the hybrids, the high activity of 3 and 6 could be due to the presence of heterocyclic and lipophilic substituents, respectively, on the Schiff base component of the hybrids.The experiment was repeated three times, with the finding reported as mean ± SD (Figs.S15 and S16).
For a compound to be a potential anticancer drug candidate it must be nontoxic to healthy cell lines.Compounds 1-6 were screened for their toxicity against MRC5 normal cell lines at various concentrations (5-100 μg/ Table 2. Synthesized hybrids of 1,2,3-triazole and chiral Schiff bases (2-6), 1 H and 13 C NMR chemical shifts of azomethine moiety, IR absorption of C = N and their CHN elemental analysis.mL) and their test results are depicted in Fig. 4. All the compounds were found to be nontoxic and exhibited cell viability (%) 51-92, at the concentration range of 5-75 μg/mL better than the standard cisplatin, which showed cell viability (%) 55-80, at the concentration range of 5-50 μg/mL.Therefore, the compounds could be viable potential candidates for the development of new anticancer drugs.The selectivity index (SI) shown in Table 4, was calculated as the ratio of the IC 50 for the normal cell line (MRC5) to the IC 50 for a respective cancerous cell line.Higher values of SI indicate greater anticancer specificity and the compounds displaying SI values higher than 3 were considered to be highly selective 45 .Some of the compounds not only had high cytotoxic activity against cancer cells but also displayed low toxicity against normal (MRC5) cells and their SI values were higher than 3.5.The SI values of compound 3 and 6 in A375 cancer cells were 3.95 and 3.85, respectively.The two compounds have high cytotoxicity to the cancer cells and low cytotoxicity to healthy cell lines.Compounds 3 and 6 were selected as potent compounds for further investigation using computational and molecular docking studies.

DFT study and chemical reactivity parameters
DFT studies were carried out on all the compounds.Their optimized ground state geometries was obtained at the B3LYP-GD3/6-311 + + G(d,p) level of theory using Gaussian16 Rev B.01 software.The frontier molecular orbitals, HOMO and LUMO were studied as well as the energy gap between the HOMO-LUMO orbitals which is indicative of the stability and reactivity of these compounds.The HOMO is a region in which electrons can be transferred to unoccupied orbitals, while LUMO is an electron-accepting spot.HOMO was found as bonding orbitals that are dispersed throughout the molecule.Several studies have shown that a lower ΔE HOMO-LUMO corresponds to more bioactivity of a molecule, and this has been used to explain the relative bioactivities of medicinal compounds 46 .Figures 5, 6   well as the energy gap between these frontier orbitals.In all the compounds the HOMO and LUMO are only pronounced on the Schiff base component of the hybrid compound.As shown in Fig. 5, for compound 1 its HOMO and LUMO are evenly distributed over the whole molecule, but the HOMO is more delocalized over the component atoms when compared to its LUMO.In contrast, the main contributions to the HOMO and LUMO of compound 2 are the atoms in its benzene ring and imine moiety.The ΔE HOMO-LUMO for compound 2 is slightly greater than 1 and this is due to the replacement of -C (H)=O group from compound 1 by -C (H)=N group in compound 2.
Figure 6 shows the HOMO and LUMO for compounds 3 and 4. In both compounds the LUMO is pronounced on the aromatic group from the parent compound and the imine moiety.The HOMO for 3 is distributed on the imine moiety and the tryptophan component, however, in compound 4 the HOMO is delocalized only on the  www.nature.com/scientificreports/aromatic group of the tyrosine component.For compound 4, the ΔE HOMO-LUMO is lower than compound 3 and this is due to the replacement of the tryptophan subunit linked to a sodium ion with a tyrosine subunit linked to two sodium ions.
As shown in Fig. 7, for compound 6 the HOMO and LUMO are evenly distributed on the aromatic group of the parent component and the imine moiety.For compound 5 the HOMO is delocalized over the whole aromatic groups of the Schiff base components (aldehyde and amine sources) and the LUMO is only pronounced on the aromatic group of the parent component and the imine moiety.For compound 5, the ΔE HOMO-LUMO is lower than compound 6 and this is due to the replacement of the phenyl alanine subunit with a histidine subunit linked to a sodium ion.
The ΔE HOMO-LUMO is 4.76, 4.68, 4.64, 4.60, 4.28 and 3.37 eV for compounds 6, 2, 1, 5, 3 and 4 respectively.So, assuming compound 1 is the parent compound, it is observed that the energy gap in compounds 6 and 2 increase slightly due to the replacement of the aldehydic moiety from compound 1 by the imine moiety linked to glutamic acid and phenyl alanine subunit, respectively.For compound 5 decreases slightly due to the replacement of the aldehydic moiety from compound 1 by the imine moiety linked to histidine subunit.The energy gap in compounds 3 and 4 decreases significantly due to the replacement of the aldehyde subunit with an imine moiety linked to tryptophan and tyrosine subunit, respectively.
The Molecular Electrostatic potential (MEP) for compounds 1, 2, 3, 4, 5, and 6 is shown in Fig. 8. MEP indicates the net electrostatic effect exerted at a point in space by the total charge distribution over a molecule.It can be used to study the reactivity of molecules towards electrophilic and nucleophilic reagents as well as their drug-receptor interactions.The different colours on the surfaces (Fig. 8) are indicative of their electrostatic potential values; it increases in the order red < orange < green < blue, where the higher electrostatic potential negative (red) regions of the MEP map are related to electrophilic attack reactivity, whereas the positive (blue) regions are related to nucleophilic attack reactivity, neutral region is represented by green colour.For compound 1, the positive (blue) regions are localized in the oxygen atoms of the carbonyl and ether and on nitrogen atoms of the 1,2,3-triazole ring.The negative (red) regions are found on the aromatic subunit linked to the 1,2,3-triazole component.For compound 2, the negative (red) regions are distributed across most of the components of the molecule and the positive (blue) regions are localized on the two oxygen atoms of the carboxylate and partially on the aromatic subunit linked to the 1,2,3-triazole component.For compounds 3 and 6, the positive (blue) regions are diffused across the whole components of the molecules and the few negative (red) regions are found on the aromatic hydrogen atoms.For compound 4, the positive (blue) regions are found in most of the components of the molecule.The negative (red) regions are localized on the two sodium ions linked to the tyrosine subunit and on the aromatic subunit linked to the 1,2,3-triazole component.For compound 5, the positive (blue) regions are distributed across the Schiff base component of the molecule and the negative (red) regions are localized on the 1,2,3-triazole, aromatic subunit linked to the triazole and on the sodium ion of the carboxylate moiety of histidine subunit.Therefore, the presence of positive (blue) and negative (red) regions on the molecules are evidence for the potential bioactivity of the compounds that could interact through its electrophilic and nucleophilic sites.

Molecular docking analysis
The docking investigation on the studied compounds as Androgen receptor modulators (PDB ID: 5t8e) and Human MIA (PDB ID: 1i1j) inhibitors to down-regulate prostate and skin cancer were executed.The adequate choice of the docking target function impacts the accuracy of the ligand positioning as well as the accuracy of the protein-ligand binding energy calculation.In this work, the protein structure that is chemically similar to the studied compounds was selected for docking study and before the execution of the docking calculation, the flexibility of the selected protein was evaluated and the proteins with appropriate flexibility was selected for docking study.Also, the method of preparation and resolution (≤ 2 Å) of the studied target which agreed with the standard before subjected to further study were considered.The inhibiting activities of the studied synthesized compounds against the studied receptors were compared with the inhibiting activity of Cisplatin against Androgen receptor modulators and Human MIA.As shown in Table 5, the calculated binding affinities for the studied compounds were higher than the reported binding affinity for the referenced compound (Cisplatin).This showed that all the studied compounds proved to be potent in inhibiting Androgen receptor modulators and human MIA than Cisplatin.More so, compound 3 and 6 has proved to be more potent in inhibiting the targets than other studied compounds.This could be confirmed via the combination of amino acid residues and types of biological interactions involved in the docking study between compound 3 and 6 in the active site of Human MIA (PDB ID: 1i1j) complexes and Androgen receptor modulators (PDB ID: 5t8e), respectively.This docking result is in good agreement with the high anticancer activity for compounds 3 and 6 observed in the experimental invitro anticancer study.The type of interactions involved in compound 3-Human MIA complexes and compound 6-Androgen receptor modulators were observed to increase the level of stability and selectivity in the active site of the targets (Figs. 9 and 10).

Absorption, distribution, metabolism, and excretion (ADME) prediction
Druglikeness evaluates whether a particular molecule is similar to the known drug or not.It is a complex balance of various properties and structural features of a compound.Lipinski's rule is widely used to determine molecular properties that are important for drug's pharmacokinetic in vivo.According to Lipinski's rule of five, a candidate molecule is more likely to be orally active if: (a) MW ≤ 500, (b) MLogP ≤ 4.15, (c) HBD ≤ 5, (d) HBA ≤ 10, and (e) the number of violations ≤ 1 47 .These parameters were calculated by the online available swissADME web tool (http:// www.swiss adme.ch/) and are presented in Table 6.Low molecular weight drug molecules (< 500) are easily transported, diffuse, and absorbed as compared to heavy molecules.The molecular weight of all the compounds were found to be less than 500.Partition coefficient or Log P is an important parameter used in rational drug design to measure molecular hydrophobicity.Hydrophilic/lipophilic nature of drug molecule affects drug absorption, bioavailability, drug-receptor interactions, metabolism of molecules, as well as their toxicity.All the Lipophilicity plays an important role in the distribution of drug after absorption in the body.All the compounds have less than 5 and 10, hydrogen bond donors and acceptors, respectively which obeys the Lipinski's rule of five.Topological polar surface area (TPSA) is closely related to the hydrogen bonding potential of a molecule and is a very good predictor of drug transport properties such as intestinal absorption, bioavailability, blood brain barrier penetration etc 48 .TPSA of all the compounds were found in the range of 87.83-126.90 and it is in the acceptable range of < 160 Å limit.Number of rotatable bonds is a simple topological parameter that measures molecular flexibility and is a good descriptor of oral bioavailability of drugs.The greater the number of rotatable bonds, the more flexible the molecule is to achieve different conformations.The number of rotatable bonds for the compounds were in the range of 9-11.The topological parameter and the number of rotatable bonds are considered to be good descriptors of the oral bioavailability of drugs 49 .
The drug under study is supposed to bind with the biological target.The biological target can be any common protein such as ion channels, enzymes, and receptors.The biological target is also known as the drug target.The predicted bioactivity scores of screened compounds as well as their comparison with the standard drug for GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor and enzyme inhibitory activity was computed using Molinspiration cheminformatics software (freely available on https:// molin spira tion.com) and are summarized in Table S4.In general, if the bioactivity score (G protein-coupled receptor (GPCR) ligand, a kinase inhibitor, ion channel modulator, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor) of the synthesized compounds is > − 0.5, then the drug is biologically active, but if the score is < − 0.5, then the drug is not active.The bioactivity scores, as provided in Table S4, showed that triazole 1 and the 1,2,3-triazole and chiral Schiff base hybrids 2-6 are active and confirmed their binding flexibilities 50 .
The Swiss ADME software provides a 'BOILED-Egg' visualization (Fig. 11) that displays two important ADME parameters: passive gastrointestinal absorption (HIA) and blood-brain barrier (BBB) access.These parameters mainly use two physicochemical descriptors: the octanol-water partition coefficient (MLogP) and the topological polar surface area (TPSA).In Fig. 11, the egg-shaped categorization plot depicts the white region, which represents the physiochemical space that favours HIA absorption, and the yolk region, which indicates properties that favour BBB permeability.Compounds 1 fall in the yolk region, indicating possible penetration of the BBB.In addition, it is likely that compounds with TPSA < 79 Å 2 and relatively lipophilic properties reach the central nervous system (CNS).However, the 'BOILED-Egg' approach is limited to passive molecules.The blue dots on the diagram indicate compounds that are likely to be removed from the CNS by p-glycoproteins, while the red dots indicate compounds that are likely to remain in the CNS.Compounds 2-6 exhibit favorable physicochemical properties with low molecular weights and TPSA values < 140 Å 2 , indicating good human intestinal absorption (HIA) 51 .
Table 5. Calculated binding affinity and interaction involved between the studied complexes.

Type of Interaction involved in the interaction
Binding Affinity (kcal/ mol)

Type of Interaction involved in the interaction
Binding Affinity (kcal/ mol)

Conclusion
The 1,2,3-triazole and chiral Schiff base hybrids 2-6 were successfully synthesized and their chemical structures were established using different spectroscopic techniques.Crystal of compound 1 was obtained and its structure was confirmed by single crystal X-ray diffraction.All compounds were evaluated for their anticancer activity against PC3 prostate cancer, skin cancer and MRC5 normal cells.All the compounds showed significant anticancer activity against the cancerous cells.Among the tested compounds 3 and 6 showed high activity for the inhibition of A375 and PC3 cancer cell lines and low toxicity for the healthy cell lines (MRC5).DFT study on the compounds proved the presence of electrophilic and nucleophilic bioactive sites for receptors.Molecular docking study proved that all the compounds are potent in inhibiting Androgen receptor modulators and human MIA than Cisplatin.The high binding affinity of compounds 3 and 6 plays a vital role for their high anticancer activity observed in the experimental invitro anticancer evaluation.Structure activity relationships (SARs) of the tested compounds is in good agreement with DFT and molecular docking studies which proved that the presence of heterocyclic and lipophilic substituent on the Schiff base component of the hybrids is the main factor for high anticancer activity.The compounds exhibited desirable physicochemical properties for druglikeness.Thus, this preliminary study could be a foundation for researchers to gain more understanding on the synthesis and anticancer activity of 1,2,3-triazole and chiral Schiff base hybrids.

Analytical
Melting points were determined using a Reichert-Jung Thermovar hot-stage microscope and are uncorrected.Infrared spectra were recorded using Tensor 27 Bruker and Perkin Elmer FT-IR spectrum BX.High resolution mass spectra (HRMS) or mass spectra (MS) were carried out on a Waters Synapt G2 instrument at University of Pretoria, South Africa.All 1 H NMR spectra were recorded on Bruker 500 MHz NMR spectrometer at ambient   The crystal was mounted on a glass fibre and used for the X-ray crystallographic analysis.The X-ray intensity data were collected on a Bruker Apex DUO 4 K CCD diffractometer area detector system, equipped with a graphite monochromator and Mo K α fine-focus sealed tube (λ = 0.71073 Å) operated at 1.5 KW power (50 kV, 30 mA).The detector was placed at 4 cm from the crystal.Crystal temperature during the data collection was kept constant at 100 (2) K, using an Oxford 700 + series cryostream cooler.

Synthesis of 2-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (1)
To solution of 2-(prop-2-yn-1-yloxy)benzaldehyde (1.35 g, 8.44 mmol) in DMF: H 2 O (100 mL.4:1) was added azidobenzene(1.21mL, 10.13 mmol) followed by the addition of CuSO 4 .5H 2 O (0.0600 mmol, 15.0 mg dissolved in 200 µL of water) and sodium ascorbate (0.880 mmol, 174 mg dissolved in 800 µL of water).The reaction mixture was stirred under reflux for 24 h.The hot solution was poured into ice-water mixture, and the precipitate formed was filtered, washed with ice-water (3 × 25 mL).The solid product was dried in vacuo at 100 °C to provide compound 1 as colourless solid.The solid product collected was then recrystallised from ethanol (20 mL, by slow evaporation) and provided compound 5 as crystalline solid 44  General procedure for the synthesis of 1,2,3-triazole and chiral Schiff bases hybrids (2-6) The reaction was carried out following the literature reported procedure 52 .To a stirred solution of sodium hydroxide (0.043 g, 1.08 mmol) in 20 mL methanol at 80 °C was added two equivalents of the amines.When the combined reaction mixture completely dissolved, compound 1 (0.100 g, 0.358 mmol) was added and the colour of the reaction solution eventually changed to yellow.The reaction was stirred for 1 h at 80 °C.After the solution was cooled down to room temperature 150 mL aliquots of diethyl ether were added.A yellow precipitate appeared.The yellow precipitate was separated from the solution, washed several times with diethyl ether and dried in vacuo.

Figure 5 .
Figure 5. Frontier molecular orbital diagram and energy values for compounds 1 and 2.

Figure 6 .
Figure 6.Frontier molecular orbital diagram and energy values for compounds 3 and 4.

Figure 7 .
Figure 7. Frontier molecular orbital diagram and energy values for compounds 5 and 6.

Figure 9 .
Figure 9. 3D and 2D structure of compound 3 in the active site of human MIA.

Figure 10 .
Figure 10.3D and 2D structure of compound 6 in the active site of Androgen receptor modulators.
temperature and are reported as chemical shift δ in units of parts per million (ppm) with reference to the solvent (2.50 ppm for DMSO-d 6 and 7.26 ppm for C(H)DCl 3 ) or TMS (0.00 ppm).Multiplicities are presented as: s (singlet); d (doublet); t (triplet); dd (doublet of doublet); and m (multiplet).Coupling constants J values are expressed in Hz and the number of protons expressed as nH.13 C NMR spectra were obtained using Bruker 500 MHz NMR spectrometer at ambient temperature.Spectra are reported as chemical shift δ in units of parts per million (ppm) with reference to the deuterated solvent (39.81 ppm for DMSO-d 6 or 77.16 ppm for CDCl 3 ).

Table 1 .
Crystal data and structure refinement for compound 1.

Table 4 .
The calculated values of the selectivity index (SI) for compounds 1-6.

Table 6 .
In silico physicochemical data for drug likeness based on the Lipinski rule (SAR).a Molar refractivity; b octanol-water partition coefficient, calculated by methodology developed by Molinspiration; c polar surface area; d number of non-hydrogen atoms; e molecular weight; f number of hydrogen-bond acceptor (O and N atoms); g number of hydrogen-bond donors (OH and NH atoms); h number of "Rule of five" violations; i number of rotable bonds; j molecular volume.